RESUMEN
Optogenetics is a modern technique which has been recently expanded to non-neuronal cell types, e.g., astrocytes, and involves targeted gene delivery of light-sensitive ion channels like Channelrhodopsin-2 (ChR2). Optogenetic regulation of astrocytic activity can be used for therapeutic intervention of several neurological disorders. Astrocytic gene delivery, viz adeno-associated viral (AAV) vectors, have proven to be robust, time-, and cost-efficient contrary to the generation of transgenic animal models. When transducing astrocytes with an AAV vector, it is imperative to perform a serotype evaluation of the AAV vector due to variability in serotype transduction efficiency depending on species, target region and construct length. Rats have been a very successful animal model for studying a variety of brain disorders, from which ChR2-based intervention of astrocytes will benefit. However, the most efficient AAV capsid serotype targeting astrocytes for ChR2 expression in the in vivo rat brain cortex has not been characterized. To address this, we have evaluated AAV serotypes 1, 5, and 8 of the vector AAV-GFAP-hChR2(H134)-mCherry targeting astrocytes in the rat brain neocortex. Results show that serotype 8 exhibits promising transduction patterns, as it has demonstrated the highest tangential and radial viral spread in the rat brain. Our research will facilitate translational research for future applications of optogenetics involving the transduction of rat brain cortical astrocytes.
Asunto(s)
Astrocitos/metabolismo , Corteza Cerebral/metabolismo , Channelrhodopsins/genética , Dependovirus/genética , Vectores Genéticos/administración & dosificación , Optogenética , Transducción Genética , Animales , Técnicas de Transferencia de Gen , Terapia Genética , Vectores Genéticos/genética , Masculino , Ratas , Ratas Wistar , Serogrupo , TransgenesRESUMEN
Astrocytes are actively involved in a neuroprotective role in the brain, which includes scavenging reactive oxygen species to minimize tissue damage. They also modulate neuroinflammation and reactive gliosis prevalent in several brain disorders like epilepsy, Alzheimer's, and Parkinson's disease. In animal models, targeted manipulation of astrocytic function via modulation of their calcium (Ca2+ ) oscillations by incorporating light-sensitive cation channels like Channelrhodopsin-2 (ChR2) offers a promising avenue in influencing the long-term progression of these disorders. However, using adult animals for Ca2+ imaging poses major challenges, including accelerated deterioration of in situ slice health and age- related changes. Additionally, optogenetic preparations necessitate usage of a red-shifted Ca2+ indicator like Rhod-2 AM to avoid overlapping light issues between ChR2 and the Ca2+ indicator during simultaneous optogenetic stimulation and imaging. In this article, we provide an experimental setting that uses live adult murine brain slices (2-5 months) from a knock-in model expressing Channelrhodopsin-2 (ChR2(C128S)) in cortical astrocytes, loaded with Rhod-2 AM to elicit robust Ca2+ response to light stimulation. We have developed and standardized a protocol for brain extraction, sectioning, Rhod-2 AM loading, maintenance of slice health, and Ca2+ imaging during light stimulation. This has been successfully applied to optogenetically control adult cortical astrocytes, which exhibit synchronous patterns of Ca2+ activity upon light stimulation, drastically different from resting spontaneous activity. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Experimental preparation, setup, slice preparation and Rhod-2 AM staining Basic Protocol 2: Image acquisition and analysis.
